This project has as its final goal to understand the structural changes in tubulin related to nucleotide hydrolysis and resulting in the dynamic nature of microtubules. Dynamic instability is an essential property of microtubule required for the progression of mitosis and other cellular functions. GTP hydrolysis in beta-tubulin causes a conformational change in the dimer that weakens lateral interaction and induces the curling of protofilaments that results in microtubule depolymerization. We will compare the structures of tubulin in two different conformations representing the high and low energy states of the protein: the conformation in a straight protofilament where the GDP-tubulin molecule is constrained in a "GTP-like" state by the polymer lattice, and the conformation in curved protofilaments where GDP-tubulin is in an unconstrained, relaxed state. We have an atomic structure of tubulin in the constrained state obtained by electron crystallography of zinc-induced sheets, as well as an 8 Angstrom resolution model of the intact microtubule. Thus the present project is concerned with obtaining the low energy structure of GDP-tubulin by cryo-electron microscopy and image reconstruction. We have been able to obtain a form of GDP-tubulin in which curved protofilaments pack into a crystalline tube amenable to helical reconstruction. Our final goal is to obtain a reconstruction of these tubes at better than 10 Angstrom resolution where individual alpha helices can be visualized. The crystal structure of tubulin will then be used to model this unconstrained conformation and to test different hypotheses linking the nucleotide site with polymerization surfaces on tubulin. A number of cellular factors regulate dynamic instability by either stabilizing microtubule structure or inducing its disassembly. In addition, a variety of antimitotic agents with anticancer properties function by binding to tubulin and disrupting normal microtubule dynamics. In both cases the ligand can be thought as operating on tubulin to enhance, modify or eliminate the effect of GTP hydrolysis. A detailed knowledge of the conformational changes triggered by GTP hydrolysis is thus essential for our understanding of these processes. Within this project we also plan to address the effect of antimitotic agents directly by investigating the effect of cryptophycin 1 on tubulin structure.